Project Summary/Abstract: Bending Primary Cilia: evidence for a structure-function relationship? ?How do cells sense their environment?? is a question that had dominated biology for decades. Until recently, that question was answered in terms of chemical sensing via transmembrane receptors. It has become increasingly evident that cells also sense and respond to mechanical forces using a variety of sensors. The primary cilium is hypothesized to be a mechanical sensor that senses fluid flow, with the proposed mechanism being ciliary bending. However, confirming or rejecting this ?ciliary hypothesis? has proved to be difficult. One reason is that fluid flow exerts shear stress on the entire cell surface, complicating experiments designed to determine the site of fluid flow sensing. The goal of this project is to use live cell imaging and optical trapping to apply a well-controlled force localized to a single experimenter-chosen cilium and determine the minimum amount of ciliary bending, due to flow, associated with increased cytosolic Ca2+, a well-established cellular read-out of flow stimulation. Broadly, this proposal will characterize, alter, and correlate cilium mechanical properties (?stiffness?, length) with the minimum amount of flow associated with increased cytosolic Ca2+ (?threshold flow?). This project is an important first step in understanding the interplay between mechanosensation and homeostasis. Information gained during the course of this project relating mechanical properties of the cilium to activation of a signaling pathway may enable new therapeutic approaches to ciliopathies.